Disease biomarkers are markers that can detect qualitative and quantitative changes in structure or concentration to diagnose diseases. Excess disease biomarkers are present in blood as a result of leakage of proteins from tissues, which is involved in the development of a disease. Accurate detection of the disease biomarkers is crucial to diagnose the disease. Recent research efforts have been directed towards the development of disease diagnostic technology through comprehensive verification of some proteins or peptides closely related to corresponding diseases rather than single biomarkers. Under these circumstances, there is a need for diagnostic technologies based on disease biomarkers suitable for quantitative multiplexed analysis.
For multiplexed analysis, nanoprobes should generate different signals without any interference between the signals and should respond sensitively to target analytes. Generally, fluorescent molecules bound to nanoprobes have been used for signal detection in quantitative measurements based on antigen-antibody reactions. However, there is a limitation in multiplexed analysis using different fluorescent molecules because broad peaks appear in the spectra of the fluorescent molecules. The type of laser light should be changed depending on the kind of substance and the problems of photobleaching and blinking may arise. Photobleaching refers to a phenomenon in which signals get weaker with the passage of time.
On the other hand, molecules have inherent Raman signals whose spectral widths are very narrow. Accordingly, labeling of target-specific binding agents with different kinds of Raman markers enables multiplexed analysis of the targets and allows the molecules to be simultaneously excited under single light irradiation, enabling rapid detection of the targets using simple measuring systems. However, there is a difficulty in detecting target analytes due to very low intensity of Raman signals. This difficulty is overcome by the use of metal nanoparticles that can increase the intensity of Raman signals, ideally by up to 14 orders of magnitude, to provide sufficient signal intensity for measurement. Numerous studies have focused on the fabrication of nanoprobes using metal nanoparticles to enhance Raman signals, thus being suitable for various applications (Cao, Y. W. C., Jin, R. C., Mirkin, C. A.: Nanoparticles with Raman spectroscopic fingerprints for DNA and RNA detection. Science 2002, 297 (5586): 1536-1540; Nie, S. M., Emery, S. R.: Probing single molecules and single nanoparticles by surface-enhanced Raman scattering. Science 1997, 275 (5303): 1102-1106; Li, J. F., Huang, Y. F., Ding, Y., Yang, Z. L., Li, S. B., Zhou, X. S., Fan, F. R., Zhang, W., Zhou, Z. Y., Wu, D. Y. et al.: Shell-isolated nanoparticle-enhanced Raman spectroscopy. Nature 2010, 464 (7287): 392-395; Kneipp, K., Kneipp, H., Itzkan, I., Dasari, R. R., Feld, M. S.: Ultrasensitive chemical analysis by Raman spectroscopy. Chem Rev 1999, 99 (10): 2957-2976; Kim, J. H., Kim, J. S., Choi, H., Lee, S. M., Jun, B. H., Yu, K. N., Kuk, E., Kim, Y. K., Jeong, D. H., Cho, M. H. et al.: Nanoparticle probes with surface enhanced Raman spectroscopic tags for cellular cancer targeting. Anal Chem 2006, 78 (19): 6967-6973; Qian, X. M., Peng, X. H., Ansari, D. O., Yin-Goen, Q., Chen, G. Z., Shin, D. M., Yang, L., Young, A. N., Wang, M. D., Nie, S. M.: In vivo tumor targeting and spectroscopic detection with surface-enhanced Raman nanoparticle tags. Nature Biotechnology 2008, 26 (1): 83-90). However, there have been some limitations in the fabrication of nanoprobes. First, sufficiently enhanced Raman signals are, in practice, difficult to obtain, unlike theoretical values. Second, signals are not uniformly increased. Third, attachment of binding agents specific to target analytes to metal nanoparticles for biomarker detection lacks directivity, making it difficult to quantitatively analyze the target analytes.